Activation of calcium-independent phospholipase A2 (CalPLA2) in the proximal tubule contributes to cell injury by the formation of toxic amphiphiles which cause profound inhibition of NaK-ATPase during ischemia. We have purified a novel 28 kDa CaIPLA 2 enzyme from rabbit kidney cortex. lmmunohistochemical studies using anti- CaIPLA 2 polyclonal antibodies have localized this protein to the proximal tubule. Using oligonucleotide probes derived from partial amino acid sequence obtained from this novel protein, we isolated a full length cDNA from rabbit kidney cortex. This cDNA (CaIPLA2-I) encodes for a protein with a similar molecular weight to the 28 kDa protein purified from kidney cortex and its deduced amino acid sequence contains the same amino acid sequence of three peptides isolated from the purified 28 kDa CaIPLA2. Furthermore, it also contains the sequence U-F-S-Q-G, which agrees with the consensus sequence surrounding active site serines, G-X-S-X-U, observed in many carboxyl esterases including lipases. Our hypothesis is that activation of CaIPLA2 in the proximal tubule contributes significantly to cell death during hypoxia/ischemic cell injury. This project seeks to determine the cellular and biochemical mechanisms by which CaIPLA2 activation leads to renal epithelial cell injury during ischemia using the molecular genetic data we gathered in preliminary studies. We propose: I. To clone and characterize CaIPLA2 genes expressed in the kidney. We will complete the characterization of a second partial cDNA clone CalPLA 2-2 isolated from rabbit kidney and a human kidney CalPLA2 cDNA. II. To determine the role of phosphorylation and calcium in the regulation of CaIPLA 2 activity. To express CAIPLA 2 protein in insect cells and determine the effects of in vitro phosphorylation on CaIPLA 2 activity. We will examine in vivo phosphorylation of CaIPLA2 in renal tubular epithelial cells, and also we will determine the role of calcium in membrane translocation and/or activation of CaIPLA 2. Phosphorylation at specific amino acid residues will be correlated with enzymatic activity. III. To establish the role of CaIPLA 2 in ischemia-induced renal epithelial cell injury. The cellular distribution of CaIPLA 2 and nephron compartmentalization during in vivo ischemia will be established and the cellular events that link activation of CaIPLA 2 and cell death during ischemic cell injury will be defined. Overall, these studies should help define the pathway(s) by which CaIPLA 2 activation leads to proximal tubule cell death during ischemia.